Dietary copper affects survival, growth, and reproduction in the sea urchin Lytechinus variegatus.
Article Type: Report
Subject: Copper in the body (Research)
Copper in the body (Properties)
Sea urchins (Research)
Sea urchins (Physiological aspects)
Sea urchins (Food and nutrition)
Animal feeding and feeds (Research)
Gonads (Research)
Authors: Powell, Mickie L.
Jones, Warren T.
Gibbs, Victoria K.
Hammer, Hugh S.
Lawrence, John M.
Fox, Joe
Lawrence, Addison L.
Watts, Stephen A.
Pub Date: 12/01/2010
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2010 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2010 Source Volume: 29 Source Issue: 4
Topic: Event Code: 310 Science & research
Product: Product Code: 2048000 Prepared Feeds; 3523860 Livestock Feeders NAICS Code: 311119 Other Animal Food Manufacturing; 333111 Farm Machinery and Equipment Manufacturing
Geographic: Geographic Scope: United States Geographic Code: 1USA United States
Accession Number: 247523225
Full Text: ABSTRACT Copper is an essential micronutrient in the diets of animals. It is a component of many enzymes involved in energy production, participates in immune function, and protects cells from free radicals. However, excessive levels in the diet can be toxic. Small (~13 g wet weight) Lyteehinus variegatus were fed formulated feeds with 12, 36, or 114 mg Cu/kg for 12 wk (levels based on established dietary levels for other marine invertebrates, supplemented as CuS[O.sub.4] x 5[H.sub.2]O). Under these experimental conditions, wet weights of individuals fed a 36-mg Cu/kg diet were slightly higher (43.2 [+ or -] 1.2 g (SEM); P = 0.069) than those fed a 12-mg Cu/kg and 114-mg Cu/kg diet (39.9 [+ or -] 1.2 and 40.3 [+ or -] 1.7 g wet weight, respectively). Ovary and gut wet weights were significantly lower (P < 0.003) in the 114-mg Cu/kg diet than the 12-mg Cu/kg and 36-mg Cu/kg diets (7.24 [+ or -] 0.75 g, 8.11 [+ or -] 0.55 g, and 4.99 [+ or -] 0.32 g ovary wet weight and 0.97 [+ or -] 0.04 g, 1.07 [+ or -] 0.06 g, and 0.83 [+ or -] 0.04 g gut wet weight for the 12-, 36-, and 114-mg Cu/kg diets, respectively). Mature gamete formation in ovary and testis was inversely correlated with dietary copper level. Acini from the ovaries and testis of urchins in the 36-mg Cu/kg and 114-mg Cu/kg diet treatments had a greater area occupied by nutrient phagocytes than urchins on the 12-mg Cu/kg diet. In diets containing low dietary copper (12 mg Cu/kg), survivorship decreased from 100% to 87%. These data suggest that dietary copper is essential for normal physiological function but can be detrimental for certain physiological processes at high levels. This information will help in the development of formulated feeds for sea urchin aquaculture.

KEY WORDS: urchin, copper, survival, growth, reproduction, Lytechinus

INTRODUCTION

As harvesting pressures continue to increase on natural sea urchin populations, most of the major fisheries have collapsed or are in a state of decline (Andrew et al. 2002). Aquaculture holds the potential to supplement growing markets for sea urchin roe (uni) around the world; however, successful culture requires an understanding of the nutritional requirements of sea urchins. This includes the requirements for both macro- and microminerals.

Of the microminerals, copper is considered an essential nutrient for aquatic organisms and is often supplemented in formulated production diets. Copper functions as a cofactor in a variety of important enzymes involved in an array of biological processes required for growth, development, maintenance, and reproduction. It is essential in many enzymes required for energy production and protection of cells from free radical damage (Lorenzo et al. 2002). In addition, copper is required for the proper function of innate immune response in many animals (Kelly et al. 1995). In mammals, dietary copper deficiency has been linked to impaired immune cell function and a decrease in the number of mononuclear immune cells (Kelly et al. 1995). Copper levels in the diet must be carefully controlled because of its toxicity at high levels to most organisms. Dietary copper requirements have been established for some commercially important aquaculture species; however, requirements among species appear to be highly variable. Reported daily copper requirements for fish are 1-4 mg Cu/kg dry mass (Bury et al. 2003). Lee and Shiau (2002) reported a dietary copper requirement for growing the black tiger shrimp, Penaeus monodon, of 15-21 mg Cu/kg in the diet. Davis et al. (1993) estimated the dietary copper requirement of Penaeus vannarnei to be 34 mg/kg. To determine whether copper supplementation of formulated diets is required for sea urchins, we examined the effect of 3 dietary copper levels on weight gain, organ production, and gamete development in the sea urchin Lytechinus variegatus.

MATERIALS AND METHODS

Collection, Culture, and Initial Measurements

Lytechinus variegatus were collected in October 2004 from Eagle Harbor in Port St. Joseph Peninsula State Park, FL (30[degrees]N, 85.5[degrees]W) and transported to the Texas Agrilife Mariculture Research Station in Port Aransas, TX. At the time of collection, gonads were of minimal size (0.07 [+ or -] 0.02 (SEM) g wet weight, n = 16). Sea urchins were held for 1 mo in 750-L tanks at 32 [+ or -] 2 ppt salinity and 22 [+ or -] I[degrees]C. Natural seawater was filtered to 0.5 gm via stratified sand filtration using a Diamond water filter (Diamond Water Conditioning, Horton, WI), and then piped to culture raceways under flow-through conditions. During this period, sea urchins were fed a maintenance ration (approximately once every 3 days) of a formulated diet (31% crude protein (Hammer et al. 2006)).

Individual L. variegatus (n = 16 per diet treatment, 13.7 [+ or -] 0.2 g wet weight, 30.1 [+ or -] 0.2 mm diameter) were haphazardly selected and blotted on a paper towel for 15 sec to remove excess water. Test diameter was measured at 2 perpendicular points across the ambitus using calipers, and individuals were weighed to the nearest milligram. A haphazard subsample of sea urchins (n = 16) was removed, weighed as described, and dissected using an incision along the outside of the peristomial membrane, located on the oral surface. The internal organs were removed and separated into the test with spines, Aristotle's lantern, gut, and gonad (ovary or testis; n = 16 for each tissue). The gut (esophagus, stomach, and intestine combined) was rinsed thoroughly in a finger bowl to remove its contents. Each of the organs was blotted on a paper towel for 15 sec to remove excess water and weighed to the nearest milligram (organ wet weight). Organs were placed onto preweighed aluminum pans and dried 48 h to a constant weight at 60[degrees]C. The dry organs were weighed, and moisture content was calculated by subtraction. These data were used to generate linear equations describing the relationship between tissue and total wet and dry weights. These equations were used to estimate the initial urchin total dry weight, as well as the initial organ wet and dry weights for individual urchins in each treatment. At the end of the study, sea urchins from each of the diet treatments were weighed and dissected as previously described.

For the diet trials, each sea urchin was placed individually into a cylindrical enclosure constructed of plastic mesh (diameter, 12 cm; height, 30 cm; open mesh, 4 mm) secured by plastic cable ties. Mesh enclosures were fitted into 11.5-cm internal diameter PVC couplings and elevated 5.5 cm above the bottom of the tank. Small cylindrical plastic spacers (approximately 0.5 cm thick) were then placed under the bottom of each coupling, allowing water circulation beneath the enclosures. Four of these plastic cylindrical enclosures were placed into a Fiberglas tank (volume, 20 L; bottom surface area, 0.07 [m.sup.2]), with each enclosure containing an individually monitored sea urchin. Water volume and depth were held constant in each tank by a central standpipe, with depth maintained below the top of the enclosures to prevent escape. Seawater was supplied to each enclosure at a rate of approximately 95 L/h. Four tanks containing 4 enclosures each (randomized within the system) were used for each diet treatment (n = 16 sea urchins per diet treatment). Fiberglas tanks were connected within a temperature-controlled semirecirculating aquaculture system containing mechanical and biological filtration, foam fractionation (protein skimmer), and ultraviolet disinfection. Seawater was exchanged in the semirecirculating systems at a rate of approximately 10% volume per day. Statistical analysis indicated that there was no difference among Fiberglas tanks; consequently, data were combined among treatment tanks.

Culture conditions were monitored daily to maintain 32 [+ or -] 2 ppt salinity, 22 [+ or -] 1[degrees]C, and 6-7 mg/L dissolved oxygen. The daily photoperiod was 12 h light/12 h dark over a 24-h period. Ammonia, nitrite, nitrate, and pH levels were determined weekly and maintained at or below 0.1 [+ or -] 0.05 ppm, 0.1 [+ or -] 0.05 ppm, 5 [+ or -] 2 ppm, and 8 [+ or -] 0.3, respectively. In the Gulf of Mexico, copper levels of natural seawater range from 0.2-0.4 ppb (Boyle et al. 1984).

Diets and Feed Preparation

Diets containing graded levels of copper sulfate (CuS[O.sub.4] x 5[H.sub.2]O; Fisher Scientific, Falls, NJ) were prepared from a base feed containing semipurified and purified ingredients (approximately 28% marine source ingredients, 34.6% plant source ingredients, 6.5% crude fat, 1.1% carotenoids, 0.7% vitamin premix, 18.9% mineral premix, 10.2% binder-antifungal-antioxidant). Diets were supplemented with CuS[O.sub.4] x 5[H.sub.2]O to obtain diets containing 12, 36, and 114 mg copper/kg, respectively. The copper levels reflect the range of copper found to be important for other invertebrates, including shrimp (Lee & Shiau 2002). A 0% copper diet was unattainable in this study, because most practical ingredients contain endogenous levels of copper; consequently, the 12-mg Cu/kg diet reflects a diet containing no supplemental copper. With the addition of each amount of supplemental CuS[O.sub.4] x 5[H.sub.2]O, an equivalent amount of acid-washed diatomaceous earth was removed (diets were isocaloric). Dry ingredients were blended within a twin-shell dry blender (Patterson-Kelley Co., East Stroudsburg, PA) for 10 min and then mixed in a Hobart mixer (model A-200, Hobart Corporation, Troy, OH) for 40 min. Deionized water (500 mL/ kg dry mix) was then added to the dry ingredients and mixed an additional 10 min to achieve a mash consistency appropriate for extrusion. Extrusion was accomplished using a meat chopper attachment (model A-200, Hobart Corporation) fitted with a 4.8-mm die. Moist feed strands were dried on wire racks in a forced-air oven at 35[degrees]C for 24 h to a moisture content of 8-10% and refrigerated at 4[degrees]C until used.

Feed Intake

A preweighed amount of feed pellets (weighed to the nearest milligram) was placed into individually labeled plastic bags and assigned to each individual urchin in each diet treatment (n = 16 plastic bags per treatment). Each sea urchin was fed a uniform-size pellet from their respective plastic bag once daily at an amount higher than could be consumed in a 24-h period (ad libitum feeding). Daily feed intake was estimated by visual quantification of the amount of uneaten pellet in each individual enclosure as suggested by Jones et al. (2010) and Hammer et al. (2010). Prior to the next feeding, uneaten food was manually removed by siphon. For each 4-wk period, the weight of the feed pellets proffered was determined, and feed intake was estimated for each individual. Although this method estimates feed intake, technical assistants were trained in feed intake evaluation prior to the experiments, and data are comparable among treatments. In addition, previous studies (Hammer et al. 2010, Jones et al. 2010) indicate that feed leaching was consistent among all diet treatments; consequently, these values represent overestimates of actual feed intake. Values were calculated based on dry weight of feed as fed as follows:

Total feed intake (grams/individual, as fed) over the 12-wk study:

Total feed proffered (g) - total estimated uneaten feed (g)

Daily feed intake (grams of food consumed per individual per day, as fed):

Total feed proffered (g) - total estimated uneaten feed (g)

Number of days Copper intake (milligrams of dry copper consumed per individual) over the 12-wk study:

[Total feed proffered (g) - total estimated uneaten feed (g)] x [copper (mg)/dry feed (g)]

Total tissue copper content:

Tissue dry weight x copper in tissue (ppm)

Growth

Test diameter and total wet weight were measured as described previously. The estimated dry matter production was calculated as follows:

Final sea urchin dry weight (g) - calculated initial sea urchin dry weight (g)

Feed and Tissue Copper-Level Determination

Treatment diets as well as sea urchin test, gonad (ovary or testis), and gut dry tissue samples (organs from 10 individuals per treatment) were analyzed for copper concentration at Texas A&M University, Corpus Christi. Samples were digested using a MARSXpress closed-vessel microwave digester (CEM Corporation, Matthews, NC). Copper concentration for each treatment feed or individual sea urchin organ was subsequently determined using a Varian SpectrAA 220Z Atomic Absorption Graphite Furnace system (Varian Corporation, Houston, TX).

Histology

One gonad lobe from each individual (n = 16 per treatment) was fixed for 24 h in Davidson's solution and then transferred to 70% ETOH after 24 h. Tissue samples were dehydrated, embedded in paraffin, sectioned at a thickness of 5 [micro]m, and stained with eosin and hematoxylin. Gonad histology was evaluated using an SMZ-1000 zoom stereomicroscope with 2x objective and DSFil color camera (Nikon, Milville, NY). Total area of individual acini (4-6 per individual) were measured, and the areas occupied by nutritive cells and developing gametes were determined using color analysis with the NIS-Elements imaging software (version 2.34, Nikon).

Statistics

Normality and homogeneity of variances were tested initially using Kolmogorov-Smirnov and Levene tests, respectively, and all data satisfied the assumptions for parametric analysis. The analyses for total wet weight and test diameter were performed using a repeated-measures model with PROC MIXED (SAS version 9.1; SAS Institute, Cary, NC). The effects of treatment group, time in weeks, and the interaction of treatment group and time were evaluated by F tests. The apriori planned comparisons of specific differences in predicted treatment means averaged over time and at each time point (0, 4, 8, 12 wk) were compared using Tukey's adjustment for multiple comparisons. For all other data, analysis of variance (ANOVA) was performed to evaluate the effect of dietary treatment. Analysis of covariance (ANCOVA) was performed to adjust for size to evaluate the direct effect of dietary treatment on organs. Wet or dry test was used as a covariant for organ wet and dry weights, respectively. Male and female gonads were analyzed separately. When the null hypothesis was rejected, Tukey's adjustment was used to compare each pair of group means. All assumptions for ANCOVA analyses were met, including linearity of covariate and dependent variable, homogeneity of variance, and homogeneity of regression. Because all results of ANCOVA were the same as ANOVA, only results for ANOVA are presented. For all analyses, P < 0.05 was considered statistically significant. Histological data were also studied by ANOVA, and Tukey's adjustment was used to compare each pair of group means to determine significance (P < 0.05) in gonad maturity based on area occupied by parietal and mature gametes and nutritive phagocytes.

RESULTS

Survival

Two of 16 urchins died at week 11 in the 12-mg Cu/kg diet treatment. These urchins developed black lesions associated with the epithelial layer. They subsequently showed a decrease in food consumption and spine loss around the lesion. Postmortem dissections of these urchins revealed large numbers of red coelomocytes in the coelomic fluid. Survival in all other treatments was 100% over the 12-wk study period.

Feed Intake

Total feed intake did not differ significantly (P [greater than or equal to] 0.05) between males and females within treatments, and data were combined for analysis. Feed intake was significantly lower (P < 0.05) for urchins fed the 36-mg Cu/kg diet for the 4-8-wk and 8-12-wk time periods (Table 1) compared with the feed intake for the 12-mg Cu/kg and 114-mg Cu/kg diets for the same time periods. Total feed intake was significantly lower (P < 0.05) for the 36-mg Cu/kg diet than the 12-mg Cu/kg and 114-mg Cu/kg diets.

Bioaccumulation

Copper was detected in all the tissues derived from all treatments and was highest in tissues of urchins fed the 114-mg Cu/kg diet (Table 2). The gut had the highest level of copper for all tissues regardless of diet. Copper was significantly higher in the gut from urchins fed the 114-mg Cu/kg diet (P < 0.05) compared with the 12-mg Cu/kg and 36-mg Cu/kg diets. The copper level in the gonad tissue did not differ significantly between the 12-mg Cu/kg and 36-mg Cu/kg diet, but was significantly higher in urchins fed the 114-mg Cu/kg diet (P < 0.05) than the 12 mg Cu/kg and 36 mg Cu/kg diets. At 12 wk, the total copper accumulated in the test was greater than that for any of the other tissues for all of the diets because of its higher percent of the body weight (Table 3). The organs from individuals in the 114-mg Cu/kg diet had the highest levels of copper accumulation compared with the 12-mg Cu/kg and 36-mg Cu/kg diets.

Weight Gain

All diets supported wet weight gain over the 12-wk period. At week 12, there were no significant differences in test diameters among the 3 dietary treatments (Table 4). Mean wet weight was significantly (P < 0.05) higher in sea urchins fed the 36-mg Cu/kg diet at weeks 8 and 12 compared with the other diet treatments. Total dry weight did not differ significantly among the treatments at either week 0 or week 12.

Organ Wet and Dry Weight

The wet weight of tests for urchins fed the 36-mg Cu/kg diet was significantly greater (P < 0.05) than that for urchins fed the 12-mg Cu/kg diet, but did not differ significantly from those fed the 114-mg Cu/kg diet (Table 5). There was no significant difference in dry weights of tests among dietary treatments. The wet weight of the lantern from urchins fed the 6-mg Cu/kg diet was significantly heavier than those from urchins fed either the 36-mg Cu/kg or 114-mg Cu/kg diets (1.29 [+ or -] 0.06 g, 1.12 [+ or -] 0.05 g, and 1.15 [+ or -] 0.03 g, respectively; ANOVA, P = 0.03); however, there was no significant difference in dry weights of the lanterns among any of the treatments (Table 5). The lantern index (dry lantern/dry test) for the 12-mg Cu/kg diet (0.097 [+ or -] 0.003) was significantly greater (P < 0.05) than that for urchins fed the 36-mg Cu/kg and 114-mg Cu/kg dietary treatments (0.083 [+ or ] 0.0033 and 0.086 [+ or -] 0.004, respectively). Wet and dry weights of guts were significantly lower for urchins fed the 114-mg Cu/kg diet (0.83 [+ or -] 0.04 g) compared with the 12-mg Cu/kg and 36-mg Cu/ kg diet (P < 0.05). Ovary wet and dry weights for urchins fed the 114-mg Cu/kg diet were significantly lower than the 12-mg Cu/ kg and 36-mg Cu/kg diet (P < 0.05). There was no significant difference in the wet or dry weights of the testes among diet treatments (Table 5). The gonad index was significantly lower (P < 0.05) for females fed the 114-mg Cu/kg diet compared with the 12-mg Cu/kg and 36-mg Cu/kg diet (Table 6), and was significantly lower (P < 0.05) for males fed the 114-mg Cu/kg diet compared with 12-mg Cu/kg diet, but did not differ significantly from those fed 36-mg Cu/kg diet.

Histology

The ratio of acinus area occupied by parietal and mature gametes to the area encompassing nutritive phagocytic cells decreased with increasing dietary copper in females and was significantly lower in the l14-mg Cu/kg diet (0.32 [+ or -] 0.04) compared with the 12-mg Cu/kg and 36-mg Cu/kg diets (0.50 [+ or -] 0.07 and 0.42 [+ or -] 0.03, respectively; Table 7). The ratio of mature gametes to nutritive phagocytes was significantly greater (P < 0.05) in males fed the 12-mg Cu/kg diet compared with the 36-mg Cu/kg and 114-mg Cu/kg diets (0.34 [+ or -] 0.03 and 0.40 [+ or -] 0.03, respectively).

DISCUSSION

Direct measures of feed intake could not be accomplished in this study because of time and labor available; thus, feed intake was estimated as described previously (Jones et al. 2010). These feed intake estimates are reasonable, as personnel were trained in feed intake evaluation, and all evaluations were consistent. It is acknowledged that these estimates may result in increased variation and increase the potential for type II error. Individual sea urchins feed intermittently and without consistency (i.e., some individuals will feed, whole or in part, immediately, whereas others may not feed for several hours, allowing nutrient leaching) (Hammer et al. 2006). Consequently, feed estimates are always confounded by leaching, and feed intake values will be overestimates of the actual amount of feed consumed. However, these estimated feed intake values are comparable among the diets in this study.

All diets supported higher rates of weight gain than those reported for wild sea urchin populations (Beddingfield & McClintock 2000). However, weight gain differed among urchins fed different levels of dietary copper. Lower average weight gain and survival in the urchins fed the 12-mg Cu/kg diet suggest that there is a copper requirement that is not met by this lower dietary copper level. Weight gain was also lower for the 114-kg Cu/mg diet, indicating a potential negative effect of higher dietary copper levels. Elevated copper levels can also have a negative impact on animal physiology. Lower production weights of the gut and ovaries from urchins receiving the 114-kg Cu/mg diet further suggest a sublethal toxic effect. Similar organs of fish and other invertebrates accumulate dietary copper (Wang & Rainbow 2000, Kamunde et al. 2002, De Schamphelaere et al. 2007).

The patterns of copper accumulation were similar to those reported in other animals. In fish and mammals, copper concentrations have been reported to be highest in the gut and liver (Kamunde & Wood 2003). Between 30-50% of the ingested copper in mammals is absorbed in the small intestines (Gaetka & Chow 2003). In trout, copper accumulation in the tissues is directly related to dietary copper levels and is found at the highest concentration in the liver (Kamunde & Wood 2003). The accumulation of copper in the whole body has also been reported in P. monodon fed different levels of dietary copper (Lee & Shiau 2002). In L. variegatus, the gut tissue contained the highest concentration of copper, but the test, because of its larger mass, stored the highest quantity of copper. Although copper levels were elevated in urchins fed higher dietary levels of copper, the levels did not always show a direct relationship to the dietary copper levels, suggesting regulation of the copper levels in some organs when consuming low to moderate levels of copper.

No mortalities were observed in urchins fed the 114-mg Cu/kg diet in this 12-wk study. The only mortalities were of urchins receiving the lowest dietary copper level, suggesting a pathological copper deficiency. The pathology of the mortalities observed in the 12-mg Cu/kg diet was consistent with that described for a Vibrio sp. bacterial infection (Masuda et al. 2004). Copper is an important component of the immune system in many animals (Kelly et al. 1995). Although the role of copper in the immune system of sea urchins is not known, we hypothesize it is similar to that found in the immune response of other organisms. In mammals, cytochrome c and copper-zinc superoxide dismutase both contain copper, and activities of cytochrome c oxides and copper-zinc superoxide dismutase decrease in rats maintained on low-copper diets. It has been hypothesized that this decrease in enzyme activity may result in an increase in free radicals and the potential for increased tissue damage (Koller et al. 1987).

High copper levels affected reproductive processes in L. variegatus. The increased copper content of the gonad tissue corresponded to reduced gamete development in both males and females. Copper affected steroidogenesis in the testes of male albino rats resulting in suppressed male reproductive activity and reduced testicular weight (Chattopadhyay et al. 1999). In male rats, 17-beta-hydroxysteroid dehydrogenase activity is inhibited by copper (Chattopadhyay et al. 1999). This same pathway for androgen production is present in L. variegatus (Wasson & Watts 2000, Wasson et al. 2000) and could be similarly affected by dietary copper. Furthermore, transition metals, such zinc and selenium (Trawick 2009, Jones et al. 2010), which are essential in small quantities for growth and health, can accumulate in the organs and impact the development of ovaries and testis, and affect copper function. Transition metals such as copper are important components of the proteins necessary for proper biological function; however, in excess, these same essential metals are potentially toxic (Bury et al. 2003). Excess copper can bind inappropriately to biologically sensitive molecules or can form free radicals (Bury et al. 2003). The growth and survival data in the current study suggest both a copper requirement and the ability of L. variegatus to regulate copper contained in the diet. Additional studies will be required to determine the copper requirement of L. variegatus during growth from juvenile to adult. However, the reduced production observed in the l l4-mg Cu/kg diet suggests that copper levels in the diet must be monitored to avoid potentially toxic effects and to preserve a functioning immune system.

ACKNOWLEDGMENTS

We thank Jeff Barry, Anthony Siccardi, Patty Waits Beasley, and the rest of the staff at the Texas AgriLife Research Mariculture Laboratory in Port Aransas, Texas, for their technical support. We also thank Dr. Renee Desmond (Department of Biostatistics) and Dr. Robert Angus for statistical consultation. This publication was supported by the National Sea Grant College Program of the U.S. Department of Commerce's National Oceanic and Atmospheric Administration under NOAA grant nos. NA16RG2258 and NA06OAR4170078, the Mississippi-Alabama Sea Grant Consortium project nos. R/ SP-9 and R/SP-15, and the University of Alabama at Binningham. Additional support was provided by the Texas Sea Grant and Texas AgriLife Research Mariculture Laboratory, and the Lindberg Foundation. The views expressed herein do not necessarily reflect the views of any of those organizations.

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MICKIE L. POWELL, (1), * WARREN T. JONES, (1) VICTORIA K. GIBBS, (1) HUGH S. HAMMER, (1) JOHN M. LAWRENCE, (2) JOE FOX, (3) ADDISON L. LAWRENCE (4) AND STEPHEN A. WATTS (1)

(1) Department of Biology, University of Alabama at Birmingham, 1300 University Boulevard, Birmingham, AL 35294-1170, (2) Department of Biology, University of South Florida, 4202 East Fowler Avenue, Tampa, FL 33620; (3) Texas A&M University System, 6300 Ocean Drive, Corpus Christi, TX 78412; (4) Texas Agrilife Research Station, 1300 Port Street, Port Aransas, TX 78373

* Corresponding author. E-mail: mpowell@uab.edu
TABLE 1.
Feed intake and copper consumption in L. variegatus fed diets
containing 12, 36, or 114 mg Cu/kg for 12 wk.

  Diets             Weeks 0-4                Weeks 4-8
(mg Cu/kg)     (g/individual/day)        (g/individual/day)

    12       0.14 [+ or -] 0.004 (a)   0.28 [+ or -] 0.01 (a)
    36       0.13 [+ or -] 0.005 (a)   0.19 [+ or -] 0.01 (b)
   114       0.14 [+ or -] 0.004 (a)   0.23 [+ or -] 0.01 (a)

                                            Total Feed
  Diets            Weeks 8-12                Consumed
(mg Cu/kg)     (g/individual/day)         (g/individual)

    12       0.29 [+ or -] 0.02 (a)   20.11 [+ or -] 0.60 (a)
    36       0.22 [+ or -] 0.01 (b)   15.80 [+ or -] 0.64 (b)
   114       0.27 [+ or -] 0.01 (a)   18.60 [+ or -] 0.48 (a)

                   Total Copper
  Diets              Consumed
(mg Cu/kg)     ([micro]g/individual)

    12        239.4 [+ or -] 7.1 (a)
    36        542.3 [+ or -] 22.l (b)
   114       2,127.0 [+ or -] 54.6 (c)

Feed intake was reported for each 4-wk period. Values represent means
of 14-16 individuals per treatment [+ or -] SEM. Different letters
are significantly different among treatments (Tukey's post hoc,
P < 0.05); n = 16 per treatment.

TABLE 2.
Copper levels in dry organs of L, variegatus fed diets
containing 12, 36, or 114 mg Cu/kg for 12 wk.

Empirical Feed              Test                    Gut
Level (mg Cu/kg)         (mg Cu/kg)              (mg Cu/kg)

       12          13.2 [+ or -] 0.61 (a)    29 [+ or -] 21 (a)
       36           9.0 [+ or -] 0.58 (a)    48 [+ or -] 2.7 (a)
      114          15.5 [+ or -] 0.86 (a)   257 [+ or -] 23 (b)

Empirical Feed             Gonad
Level (mg Cu/kg)        (mg Cu/kg)

       12          12.5 [+ or -] 0.4 (a)
       36          12.5 [+ or -] 0.5 (a)
      114          28.8 [+ or -] 7.5 (b)

Values are means [+ or -] SEM. Treatments with different letters are
significantly different within rows (Tukey's post hoc, P < 0.05); n
= 10 individuals per treatment.

TABLE 3.
Total copper amounts in dry organs of L. variegatus fed diets
containing 12, 36, or 114 mg Cu/kg for 12 wk.

Empirical Feed             Test                    Gut
Level (mg Cu/kg)          (mg Cu)                (mg Cu)

       12           99 [+ or -] 6.3 (a)     6 [+ or -] 0.47 (a)
       36           70 [+ or -] 3.7 (b)    12 [+ or -] 2.1 (b)
      114          117 [+ or -] 10.8 (c)   56 [+ or -] 6.7 (c)

Empirical Feed            Gonad
Level (mg Cu/kg)         (mg Cu)

       12          2.7 [+ or -] 4.5 (a)
       36          2.6 [+ or -] 2.6 (a)
      114           62 [+ or -] 216

Values are means t SEM. For each time period, treatments with different
letters are significantly different (Tukey's post hoc, P < 0.05).
(n = 10 individuals per treatment).

TABLE 4.
Growth measurements of L. variegatus fed diets containing 12, 36,
or 114 mg Cu/kg for 12 wk.

                       Diet            Week 0

Diameter (mm)           12     31.6 [+ or -] 0.26 (a)
                        36     31.3 [+ or -] 0.21 (a)
                       114     32.0 [+ or -] 0.27 (a)
Total wet weight (g)    12    15.97 [+ or -] 0.44 (aa)
                        36    15.13 [+ or -] 0.21 (aa)
                       114    16.34 [+ or -] 0.35 (aa)
Total dry weight (g)    12     3.78 [+ or -] 0.09 (aa)
                        36     3.60 [+ or -] 0.4l (aa)
                       114     3.85 [+ or -] 0.07 (aa)

                       Diet           Week 4

Diameter (mm)           12     35.6 [+ or -] 0.25 (a)
                        36     34.8 [+ or -] 0.24 (a)
                       114     35.3 [+ or -] 0.29 (a)
Total wet weight (g)    12    23.78 [+ or -] 0.57 (a)
                        36    23.22 [+ or -] 0.51 (a)
                       114    23.51 [+ or -] 0.60 (a)
Total dry weight (g)    12               *
                        36               *
                       114               *

                       Diet           Week 8

Diameter (mm)           12     40.0 [+ or -] 0.33 (a)
                        36     39.7 [+ or -] 0.32 (a)
                       114     39.0 [+ or -] 0.52 (a)
Total wet weight (g)    12    32.94 [+ or -] 0.86 (a)
                        36    34.93 [+ or -] 0.82 (b)
                       114    31.97 [+ or -] 1.20 (a)
Total dry weight (g)    12               *
                        36               *
                       114               *

                       Diet           Week 12

Diameter (mm)           12     42.5 [+ or -] 0.41 (a)
                        36     43.2 [+ or -] 0.39 (a)
                       114     42.4 [+ or -] 0.61 (a)
Total wet weight (g)    12    39.90 [+ or -] l.40 (a)
                        36    43.24 [+ or -] 1.18 (b)
                       114    40.25 [+ or -] 1.70 (a)
Total dry weight (g)    12     11.3 [+ or -] 0.50 (a)
                        36     11.3 [+ or -] 0.50 (a)
                       114     11.0 [+ or -] 0.50 (a)

For each dependent variable at each time period, treatments with
different letters are significantly different within rows (PROC MIXED,
SAS 9.12; P < 0.05, n = 16 individuals per treatment 36 and 12 mg
Cu/kg; n = 14 individuals in treatment 12 mg Cu/kg).

* No urchins dissected at these time points.

TABLE 5.
Mean organ wet and dry weights [+ or -] SEM of L. variegatus fed
diets containing 12, 36, or 114 mg Cu/kg for 12 wk.

                           mg Cu /
                           kg diet         Week 0

Test      Wet weight (g)      12     6.71 [+ or -] 0.20
                              36     6.35 [+ or -] 0.09
                             114     6.88 [+ or -] 0.16
          Dry weight (g)      12     3.35 [+ or -] 0.09
                              36     3.18 [+ or -] 0.04
                             114     3.42 [+ or -] 0.07
Lantern   Wet weight (g)      12     1.17 [+ or -] 0.10
                              36     0.98 [+ or -] 0.05
                             114     1.26 [+ or -] 0.08
          Dry weight (g)      12     0.51 [+ or -] 0.03
                              36     0.46 [+ or -] 0.01
                             114     0.54 [+ or -] 0.02
Gut       Wet weight (g)      12     0.34 [+ or -] 0.02
                              36     0.30 [+ or -] 0.01
                             114     0.35 [+ or -] 0.02
          Dry weight (g)      12     0.07 [+ or -] 0.00
                              36     0.06 [+ or -] 0.00
                             114     0.07 [+ or -] 0.00
Ovary     Wet weight (g)      12     1.06 [+ or -] 0.20
                              36     0.69 [+ or -] 0.09
                             114     1.23 [+ or -] 0.16
          Dry weight (g)      12     0.34 [+ or -] 0.06
                              36     0.22 [+ or -] 0.03
                             114     0.39 [+ or -] 0.05
Testis    Wet weight (g)      12     1.06 [+ or -] 0.20
                              36     0.69 [+ or -] 0.09
                             114     1.23 [+ or -] 0.16
          Dry weight (g)      12     0.34 [+ or -] 0.06
                              36     0.22 [+ or -] 0.03
                             114     0.39 [+ or -] 0.05

                           mg Cu /
                           kg diet        Week 12         ANOVA *

Test      Wet weight (g)      12     14.3 [+ or -] 0.43      a
                              36     16.4 [+ or -] 0.60      b
                             114     15.1 [+ or -] 0.56     ab
          Dry weight (g)      12     7.35 [+ or -] 0.24      a
                              36     8.01 [+ or -] 0.26      a
                             114     7.71 [+ or -] 0.30      a
Lantern   Wet weight (g)      12     1.29 [+ or -] 0.06      a
                              36     1.12 [+ or -] 0.05      b
                             114     1.15 [+ or -] 0.03      6
          Dry weight (g)      12     0.71 [+ or -] 0.03      a
                              36     0.66 [+ or -] 0.02      a
                             114     0.65 [+ or -] 0.02      a
Gut       Wet weight (g)      12     0.97 [+ or -] 0.04      a
                              36     1.07 [+ or -] 0.06      a
                             114     0.83 [+ or -] 0.04      b
          Dry weight (g)      12     0.22 [+ or -] 0.01      a
                              36     0.25 [+ or -] 0.02      a
                             114     0.18 [+ or -] 0.01      b
Ovary     Wet weight (g)      12     7.24 [+ or -] 0.75      a
                              36     8.11 [+ or -] 0.55      a
                             114     4.99 [+ or -] 0.32      b
          Dry weight (g)      12     1.95 [+ or -] 0.24      a
                              36     2.25 [+ or -] 0.19      a
                             114     1.51 [+ or -] 0.12      b
Testis    Wet weight (g)      12     6.44 [+ or -] 0.50      a
                              36     6.08 [+ or -] 0.60      a
                             114     5.03 [+ or -] 0.62      a
          Dry weight (g)      12     1.97 [+ or -] 0.15      a
                              36     1.91 [+ or -] 0.22      a
                             114     1.64 [+ or -] 0.21      a

* For the effect of dietary treatment on organs, ANOVA results did
not differ from those obtained when data were analyzed by ANCOVA
using the wet or dry test as a covariant for organ wet and dry
weights. Therefore, only results from ANOVA are presented.
Treatments with different letters are significantly different within
rows for each tissue and weight.

n = 16 individuals per treatment 36 and 12 mg Cu/kg; n = 14
individuals in treatment 12 mg Cu/kg.

TABLE 6.
Gonad index of female and male L. variegatus [+ or -] SEM fed
diets containing 12, 36, or 114 mg Cu/kg at 12 wk.

   Diet                    Female
(mg Cu/ kg)         (gonad wt/total wt)

     12       18.32 [+ or -] 1.75 (a) (n = 6)
     36       19.39 [+ or -] 0.96 (a) (n = 7)
    114       14.18 [+ or -] 0.98 (b) (n = 9)

   Diet                     Male
(mg Cu/ kg)         (gonad wt /total wt)

     12       16.11 [+ or -] 1.08 (a) (n = 8)
     36       13.62 [+ or -] 1.03 (a) (b)
(n 9
    114       10.93 [+ or -] 1.89 (b) (n = 7)

Different letters within sex are significantly different
(Tukey's post hoc, P < 0.05).

TABLE 7.
The ratio of acini area occupied by parietal and mature
gametes to the area encompassing nutritive phagocytic cells in
gonad acini in L. variegatus.

                      12 mg Cu/kg              36 mg Cu/kg

Males (testis)   0.67 [+ or -] 0.08 (a)   0.34 [+ or -] 0.03 (a)
                        (n = 8)                  (n = 9)
Females          0.50 [+ or -] 0.07 (a)   0.42 [+ or -] 0.03 (a)
  (ovary)               (n = 6)                  (n = 7)

                      114 mg Cu/kg

Males (testis)   0.40 [+ or -] 0.03 (b)
                        (n = 7)
Females          0.32 [+ or -] 0.04 (b)
  (ovary)               (n = 9)

Different letters within sex are significantly different (Tukey's
post hoc, P < 0.05).
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